The CTIF provides Hillman researchers with access to state-of-the-art computer-aided imaging tools, including all ultrastructural electron microscopy (transmission electron microscopy, scanning electron microscopy, immuno-electron SEM and TEM), light and fluorescence microscopy (macro dissecting light and fluorescence, epi-fluorescence, confocal scanning and multi-photon imaging), live cell microscopy (transmitted light and fluorescence), and fluorescence specialties like FRET, spectral analysis, ratiometric imaging, and super-resolution microscopy (structured illumination microscopy [SIM] and stochastic optical reconstruction microscopy [STORM]) technologies. Also critical to data processing, a wide range of image analysis software and technical assistance is available to investigators. Beyond technical resources, the CTIF provides a highly specialized group of personnel with expertise in various aspects of optical imaging.
Light Microscopy Services
The CTIF provides light microscopy services using a variety of standard histological or optical contrasting methods, such as differential interference contrast (DIC) or phase contrast microscopy. The CTIF also provides a full spectrum of fluorescent microscopic methods. Three major techniques, all of which allow multicolor analyses of tissues and cells, are employed. They include:
- Confocal and Multi-Photon Laser Scanning Microscopy These techniques “optically section” material and allow visualization of fluorochromes within tissues and cells with exquisite spatial resolution. In addition, quantitative measurements of fluorescence intensity may be made, and serial sections may be rendered in three dimensions, allowing determination of spatial distributions of cellular label. These technologies are used extensively by Hillman members. Currently, the facility provides access to multiple confocal systems and two multi-photon systems. In addition to several benchtop single-photon systems, CTIF provides access to handheld confocal instruments, which have proven very useful for imaging gene delivery and expression in living animals.
- Fluorescence Microscopy Through standard fluorescence or brightfield microscopy, highly sensitive color camera systems, and computer image processing, the CTIF provides Hillman researchers with access to qualitative and quantitative microscopic imaging tools. These tools support assessment of tumors and penetrating cell types, providing quantitative assessment of a wide variety of parameters that include: tumor size, number and size of micro-metastases, and degree of penetration of cells into tumors. Currently, the facility provides access to four of these systems for Hillman use. In each case, the instruments are equipped with fluorescence and DIC imaging capabilities, and all image collection is performed using high resolution, cooled charge-coupled device (CCD) cameras.
- Multicolor Fluorescence Microscopy, Live Cell Microscopy, and Ratio Imaging Live cell imaging has become an essential service provided by the CTIF to Hillman investigators. This technology, when combined with multimode, multiparameter fluorescence imaging, allows molecular events to be studied in living cells in situ. At present, the CTIF has five instruments available to researchers. Apart from live cell imaging, these instruments have been expanded to allow confocal imaging, micro injection, and total internal reflection fluorescence (TIRF) imaging. These instruments are used to study various aspects of cell/cell interactions, antigen transfer, tumor cell killing, ratiometric imaging, and signal transduction.
Electron Microscopy Services
The facility is equipped with both transmission and scanning electron microscopes, as well as all necessary support equipment. Services provided include:
- standard transmission electron microscopy
- scanning electron microscopy
- thin frozen section immuno-electron microscopy
- electron microscopic autoradiography
- freeze fracture electron microscopy
These services are commonly used as an essential extension of the light microscopy services, for example, to localize an antigen with higher resolution than is available by light microscopy, to provide greater structural resolution of cells and tissues, or to provide structural studies of relevant subcellular species.
Computer-Aided Morphometry and Image Analyses
An important facet of the services provided by the CTIF is the design and implementation of image processing and morphometric analysis algorithms. These analyses are generally performed on 2, 3, or 4 dimensional digital data sets collected using the microscopes within the facility. Commonly, the facility staff will develop algorithms for feature extraction and quantitation following discussions with the Biostatistics Facility. This ensures optimal experimental design and statistical utility.